Erk7 and erk8, novel diagnostic markers for cancer

ABSTRACT

The present invention is directed in part to the discovery of a novel signal transduction pathway that regulates estrogen responsiveness. Human extracellular signal-regulated kinase 8 (ERK8) has been discovered by applicants to preferentially enhance the destruction of ERα, and loss of ERK8 is correlated with breast cancer progression. Thus monitoring the expression of ERK8 can be used as a diagnostic and therapeutic indicator of cancer and cancer progression.

BACKGROUND

Estrogen receptor alpha (ERα) belongs to the superfamily ofligand-activated transcription factors. This superfamily shares a commonmodular structure, which consists of an N-terminal region, a DNA-bindingdomain and a ligand-binding domain. ERα regulates the expression ofgenes involved in growth and development. The cellular response toestrogens in vivo is ERα-limited and a key mechanism in regulating ERαconcentration is receptor degradation. In response to estradiol, therate of ERα degradation through ubiquitination and the 26S proteasomepathway is increased by an unknown mechanism. The 26S proteasome pathwayis the major pathway of regulated proteolysis in eukaryotes and isresponsible for the destruction of ubiquitinated substrates.

Parallel studies on other members of the nuclear receptor superfamilyhave suggested a role for mitogen-activated protein kinase (MAPK(alsoreferred to as extracellular signal regulated kinase (ERK)1/2)) inregulating receptor turnover. For example, MAPK(ERK1/2) phosphorylationof Ser-294 in the human progesterone receptor (PR) increases the PRdegradation rate. MAPK(ERK1/2) is also known to phosphorylate ERα, andtherefore whether MAPK(ERK1/2) enhances ERα turnover was investigated.

MAPK(ERK1/2) belongs to a kinase subgroup the activity of which isregulated by phosphorylation/dephosphorylation of a threonine andtyrosine residue present in a Thr-Glu-Tyr (TEY) motif within theactivation loop. Other members of this subgroup include ERK5, ERK7 andERK8. ERK8 is the human functional homolog of rodent ERK7. ERK7 and ERK8(ERK7/8) differs from the other members of the TEY subgroup in multipleways. At least 50% of ectopically expressed ERK7/8 is active in growthfactor-free conditions, while MAPK(ERK1/2) and ERK5 require growthfactor stimulation for activation. Furthermore, MAPK(ERK1/2) must beco-expressed with a constitutively active MAPK kinase(MEK) in order forphosphorylation of the TEY motif in bacterial expression systems. Incontrast, the TEY motif of ERK7/8 is substantially phosphorylated inbacteria, suggesting that primary mechanism of ERK7/8 activation is byautophosphorylation. In addition, treatment of cells expressingMAPK(ERK1/2) with okadaic acid, a protein phosphatase 2A (PP2A)inhibitor, increases the amount of TEY phosphorylated MAPK(ERK1/2).However, okadaic acid has no effect on ERK7/8 TEY phosphorylation.Therefore, both activation and inactivation of ERK7/8 are distinct fromother members of the TEY subgroup.

The substrate specificity of ERK7/8 differs from that of MAPK(ERK1/2).MAPK(ERK1/2) is able to phosphorylate a variety of substrates includingc-Jun and Elk-1 in-vitro. However, ERK7/8 is unable to phosphorylatethese substrates. The cellular localization of ERK7/8 is not regulatedin the same manner as MAPK(ERK1/2). MAPK(ERK1/2) and ERK5 require growthfactor activation for translocation to the nucleus, while inoverexpression experiments ERK7/8 appears to be constitutively nuclear.ERK7/8 also possesses a unique C-terminal tail, which is absent inMAPK(ERK1/2) and shares no similarity with the C-terminal tail of ERK5.The C-terminal tail is required for nuclear localization of ERK7/8 andmay contain regions important for protein-protein interactions. Insummary, ERK7/8 is unique from other TEY family members in regulation ofactivation, substrate specificity, and nuclear localization.

ERK7/8 differs substantially from not only MAPK family members, but alsoall other known kinases. A unique identifier of ERK7/8 is the presenceof a glutamine (Q) at position 139 in subdomain VIB of the kinase domainin ERK7 (position 138 in ERK8). This domain is essential for thecatalytic activity of kinases. A search of the Protein Kinase Databaserevealed that there are no other kinases in the human kinome thatcontain a polar residue at this position. Molecular modeling of theequivalent residue in the crystal structure of ERK2 indicates that aglutamine at this position may form three hydrogen bonds with residuesin the catalytic domain of ERK7/8. One of the predicted residues thatmay become hydrogen bonded is aspartate 138 in ERK7 (aspartate 137 inERK8), a residue responsible for co-ordination of the substrate forphosphotransfer. Thus Q139 in ERK7 (Q138 in ERK8) may significantlyalter the biological properties of ERK 7/8. In in-vitro studies usingmyelin basic protein as a substrate, autophosphorylated ERK7/8 isapproximately 1000× less active than activated MAPK(ERK1/2). It ispossible that Q139 may play a role in limiting kinase activity ofERK7/8. Interestingly, mutation of Q139 to leucine, a hydrophobicresidue, enhances ERK7/8 TEY phosphorylation in cell-based studies.

ERα is believed to play a key role during the development of breast andendometrial cancers. It has been reported that an up-regulation ofexpression levels of ERα occurs during the development of intraductalcarcinomas from normal mammary glands, and a decrease in theirexpression levels occurs during the progression of breast cancer. Moreparticularly, loss of ERα is associated with aggressive breast tumorsand poor clinical outcome.

Very little is known about ERK7, and its biological function has notbeen established. As reported herein ERK7/8 preferentially enhances thedestruction of ERα but not the related androgen receptor. Other proteinkinases closely related to ERK7/8 do not enhance ERα turnover, and ERK7kinase activity is required for its effect on ERα. In human breastcells, a dominant-negative ERK7 mutant decreased the rate of endogenousERα degradation >4-fold in the presence of hormone, and potentiatedestrogen responsiveness. ERK7 targets the ERα ligand-binding domain fordestruction by enhancing its ubiquitination. As described herein, lossof ERK7/8 has now been correlated with breast cancer progression, andall ERα-positive breast tumors tested had decreased ERK7/8 expressioncompared to normal breast tissue.

As reported herein, the present studies have revealed the existence of anew signaling pathway impinging on the 26S proteasome machinery, inwhich ERK7/8 regulates hormone responsiveness in breast cells bycontrolling the rate of ERα degradation. Furthermore, the loss of thispathway appears to be correlated to the development of breast cancer.

SUMMARY OF VARIOUS EMBODIMENTS

In accordance with one embodiment of the invention a method ofdiagnosing the presence of an estrogen responsive cancer in a patientand determining a prognosis for the patient is provided. The methodcomprises the step of measuring ERK8 levels in the cells of a biologicalsample obtained from said patient (i.e. a biopsy), and determining ifthe ERK8 levels of the biological sample are significantly lower thanthose detected in non-cancerous cells (either from that patient or frompopulation data), wherein significantly lower ERK8 levels indicates thepresence of cancer cells in said patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: is a graph representing the determination of the K_(M) of ATPfor bacterially expressed ERK7. Purified ERK7 (5 nM) was incubated inthe presence of increasing ATP concentrations. The concentration of ATPsupporting maximal velocity of the kinase reaction is 1 mM. Theconcentration of ATP supporting half-maximal velocity (K_(M)) is ˜100μM. Background is the measured signal from reactions carried out in thepresence of 200 mM EDTA, which eliminates kinase activity. Thebackground has not been subtracted from the ERK7 signal. Phosphorylationof the immobilized GST-MBPtide was detected using the HRP-ELISA asdescribed in Example 1.

FIG. 2: is a bar graph representing the activity of recombinant ERK7 andERK8 and immunoprecipitated ERK7 as measured by phosphorylation ofGST-MBPtide. Purified, bacterially-expressed ERK7 and ERK8 (5 nM) andERK7 immunoprecipitated from approximately 10% of a confluent 15 cmtissue culture plate was incubated in the presence of 1 mM ATP. Thebackground values determined in the presence of 200 mM EDTA have beensubtracted. Phosphorylation of the immobilized GST-MBPtide was detectedusing the HRP-ELISA as described in Example 1.

FIG. 3: demonstrates the alignment of rat ERK7 (SEQ ID NO: 2), mouseERK7 (SEQ ID NO: 3) and human ERK8 (SEQ ID NO: 1). GenBank sequences forrat ERK7(Acc# AF078798), mouse ERK7(Acc# BC48042), and human ERK8(Acc#AY065978) were aligned using EMBL-EBI Clustal W multiple sequencealignment program at http://www.ebi.ac.uk/clustalw/. Results arepresented as a similarity chart where “*” represents identical residues,“:” represents highly similar residues, “.” represents less similarresidues, and gaps represent no similarity.

FIG. 4: is a bar graph representing the expression levels of ERK8 invarious breast cancers wherein the data has been normalized to the levelobserved in normal breast tissue.

FIG. 5 is a bar graph representing the expression levels of ERK8 invarious breast cancers in which the grade of tumor was clearly known.

FIG. 6 represents a photograph of a Western blot demonstrating that theanti-ERK7 antibody recognizes recombinant human ERK8. Recombinant humanERK8 containing an N-terminal MYC tag and a C-terminal His tag waspurified from bacteria, electrophoresed, and immunoblotted with theindicated antibody. The pTEY antibody is an antibody specific to thedually-phosphorylated TEY motif The lower molecular weight band presentin the anti-pTEY blot is a degradation product.

FIG. 7 represents a photograph of a Western blot demonstrating thatshort interfering RNA reduces the detectable signal generated by theanti-ERK7 antibody. Human MCF10A cells, which contain endogenous ERK8,were transfected with control short-interfering(si) RNA, ERK8-specificsiRNA, or RSK2-specific siRNA. The cells were serum-starved for 24 hrsand then lysed in boiling sample buffer. Lysates containing equalamounts of total protein were electrophoresed and immunoblotted with theindicated antibody. Detection of equivalent amounts of total ERK1/2,recognized by the anti-pan ERK antibody, indicates that the samples werecorrectly normalized.

FIG. 8 is a bar graph representing data demonstrating that active ERK7enhances ERα destruction. BHK cells were co-transfected with vectorsencoding ERα and either the indicated kinase (ERK7 or K43A-ERK7) orvector control (V). The transfected cells were serum-starved and treatedwith +/−10 nM estradiol (E2) for 6 hr before the addition of boilingsample buffer. Equal amounts of total protein were electrophoresed andimmunoblotted and the relative amounts of ERα were determined from theimmunoblots by densitometry. The data are expressed as the % of ERαdivided by ERα in the vector control in the absence of E2. Themeans±standard error (S.E.) are shown for n=8. *P<0.05 and **P<0.005(Student's t-test) obtained by comparing ERα levels with co-expressedERK7 to those obtained with the appropriate vector control.

FIGS. 9A & 9B represent Western blots demonstrating that ERK7specifically enhances the degradation of ERα. BHK cells wereco-transfected with vectors encoding HA-SF1 and HA-K43A-ERK7 or HA-ERK7or vector control (V). The serum-starved, transfected cells were lysed,and equal amounts of protein electrophoresed and immunoblotted, with theresults being shown in FIG. 9A. BHK cells were also co-transfected withvectors encoding ERα or HA-AR and HA-ERK7 or MYC-ERK7 or vector control(V) and the transfected cells were then aliquoted and pre-treated with50 mM cycloheximide for 2 h before the addition of 10 nM E2 or 100 nMdihydroxytestosterone (DHT). Thereafter, at various times after ligandaddition the cells were lysed and immunoblotted, with the results beingshown in FIG. 9B.

FIG. 10A & 10B represent Western blots demonstrating ERK expression inhuman breast cells. FIG. 10A represents equivalent amounts of lysatefrom serum-starved MCF-10A, MCF-7 and MDA-MB-231 cells electrophoresed,immunoblotted and probed with the indicated labeled antibodies. FIG. 10Brepresents the amount of ERK7 and ERα present in normal human breasttissue, benign tumor tissue and breast cancer tissue. The various tissuesamples were solubilized, normalized for Ran expression, electrophoresedand immunoblotted. The tumor grade was obtained from the athologist'sreport. ERα positive samples are indicated by an *.

FIG. 11 is a graph demonstrating the effects of K43A-ERK7 onERα-mediated proliferation growth in the presence of vehicle (□) or ICI182,780 (o), a pure estrogen antagonist. MCF-7 stably transfected withK43A-ERK7 were treated +/−1 mM ICI 182,780. At various times the cellswere lysed and growth determined. ERα-dependent growth was obtained bysubtracting the growth in ICI 182,780 from the growth obtained invehicle control. The results are taken from two experiments in whicheach time point was determined in triplicate and from two independentlines. *P<0.05 (Student's t-test) obtained by comparing the responseobtained with the stables expressing HA-K43A-ERK7 to the appropriatelytreated control.

DETAILED DESCRIPTION OF EMBODIMENTS

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein, the term “treating” includes alleviating the symptomsassociated with a specific disorder or condition and/or preventing oreliminating said symptoms. For example, treating cancer includespreventing or slowing the growth and/or division of cancer cells as wellas killing cancer cells.

As used herein, the term “linkage” refers to the connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding andhydrophobic/hydrophilic interactions.

A “detectable marker” is an atom or molecule that permits the specificdetection of a molecule comprising the marker in the presence of similarmolecules without a marker. Markers include, for example colored beads,radioactive isotopes, antigenic determinants, enzymes, nucleic acidsavailable for hybridization, chromophors, fluorophors, chemiluminescentmolecules, electrochemically detectable molecules and molecules thatprovide for altered fluorescence-polarization or alteredlight-scattering.

As used herein the term “solid support” relates to a solvent insolublesubstrate that is capable of forming linkages (preferably covalentbonds) with various compounds. The support can be either biological innature, such as, without limitation, a cell or bacteriophage particle,or synthetic, such as, without limitation, an acrylamide derivative,agarose, cellulose, nylon, silica, or magnetized particles.

As used herein the term “magnetic particles” refers to particles thatare responsive to a magnetic field.

As used herein, the term “antibody” refers to a polyclonal or monoclonalantibody or a binding fragment thereof such as Fab, F(ab′)2 and Fvfragments.

As used herein, the term “ERK7/8” refers to generically to either thehuman, rat or mouse extracellular signal-regulated kinases of SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and fragments thereof.

As used herein, the term “ERK8” refers to the human extracellularsignal-regulated kinase 8 polypeptide of SEQ ID NO: 1 and fragmentsthereof.

As used herein, “ERK7/8 levels” is a generic term that refers to eitherthe concentration of ERK7/8 protein or RNA, or ERK7/8 kinase activity,or any combination thereof.

As used herein, the term “ERK8 antibody” refers to an antibody thatspecifically binds to the amino acid sequence of SEQ ID NO: 1.

As used herein, “biological sample” means any biological tissue, fluid,serum, or biopsy sample taken from an individual.

An “estrogen responsive” cancer or neoplastic cell is a cell that willincrease cellular transcription, metabolism, growth or proliferationupon exposure to estrogen.

The term “neoplastic cells” as used herein refers to cells that resultfrom abnormal new growth. A neoplastic cell may be malignant or benignand includes both blood cancers and solid tumor cells.

As used herein, the term “tumor” refers to an abnormal mass orpopulation of cells that result from excessive cell division, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues. A “tumor” is further defined as two or more neoplastic cells.

EMBODIMENTS

The present invention is directed in part to the discovery of a novelsignal transduction pathway that regulates estrogen responsiveness.Human extracellular signal-regulated kinase 8 (ERK8; see SEQ ID NO: 1)was identified based on the ability of two different antibodies torecognize a protein of a MW similar to that predicted for rat ERK7 (SEQID NO: 2). Accordingly, ERK8 has been identified as the human functionalequivalent of the mouse and rat ERK7 polypeptides. A comparison of themouse and rat ERK7 and human ERK8 sequences is provided in FIG. 3 andreveals a high degree of sequence similarity between the threepolypeptides. As reported herein ERK7/8 has been discovered topreferentially enhance the destruction of ERα but not the relatedandrogen receptor. Furthermore, other protein kinases closely related toERK7/8 do not enhance ERα turnover, and ERK7/8 kinase activity isrequired for its effect on ERα.

Further support for the physiological importance of ERK7/8 is providedby applicants' observations that ERK8 is expressed in human breasttissue, an estrogen target, and that there is a decrease in ERK8expression during breast cancer progression. Furthermore, in all tumorsamples that are ERα positive the expression of ERK8 has been lost. Thisinverse correlation between ERα and ERK8 expression levels was alsoobserved in human breast cell lines.

In addition it is noted that a dominant negative ERK7 mutant is able toregulate hormone responsiveness in breast cells as measured bytranscription and proliferation. A dominant negative ERK7 mutant wasable to enhance ERα-mediated transcription to the same extent astreatment of wild type tissues with the proteasome inhibitor, MG132,which argues that endogenous ERK7 is the predominant pathway of ERαdegradation in breast cells. These results (see Example 1 for details)support a model in which ERK7/8 enhances hormone-dependent ERαdestruction through the 26S proteasome pathway by increasing ERαubiquitination. This model is supported by observations that a dominantnegative ERK7 mutant preferentially inhibits degradation of thehormone-bound compared to unbound ERα. ERK7 enhances the level ofubiquitination of the ligand-binding domain and this domain is requiredfor ERK7/8-mediated destruction. In response to hormone binding it isknown that the ligand-binding domain undergoes a conformational changeand presumably this conformational change targets hormone-bound ERα fordestruction by ERK7/8.

It has been previously reported that, in response to hormone, thephosphorylation of Ser-118 increases. ERK7/8 enhances Ser-118phosphorylation in vivo but this phosphorylation is not important forERK7/8-mediated degradation because ERK7/8 is able to enhance the rateof degradation of ERα and S118-ERα to the same extent. No evidence hasbeen reported that mutation of other putative ERK7/8 phosphorylationsites influenced ERK7/8-mediated ERα degradation. It seems unlikely thatphosphorylation of the ligand-binding domain plays a role in regulatingturnover because no enhanced phosphorylation in the ligand-bindingdomain was observed using (³²P)-orthophosphate labeling andmicrosequencing of the radiolabeled ERα peptides in the absence orpresence of lactacystin. Accordingly, applicants believe that ERK7/8 isimportant in maintaining the homeostasis of a normal breast cell andthat with high frequency ERK7/8 is lost at an early step in breastcancer progression. Perhaps during tumor progression ERK7/8 downstreameffectors have been inappropriately activated, resulting in reduced ERαlevels even in the absence of ERK7/8.

Since the loss of ERK7/8 has been discovered to be correlated withbreast cancer progression, one aspect of the present invention isdirected to measuring endogenous ERK7/8 levels as a diagnostic andtherapeutic indicator of cancer and cancer progression. In oneembodiment a method of diagnosing the presence of an estrogen responsivecancer in a patient is provided, wherein the method comprises the stepof determining ERK7/8 levels in the cells of a biological sampleobtained from said patient; and determining whether the ERK7/8 levelsare below a threshold level. The expression levels of ERK8 protein invarious breast cancer cells has been quantitated and normalized to theERK8 protein levels observed in normal breast tissue (see FIG. 4). Asshown in FIG. 4, relative to non-cancerous and benign tumors sevendifferent cancers have significant reduced ERK8 protein levels.

In accordance with one embodiment a method of early detection ofestrogen receptor associated cancers, and more particularly breastcancer, in patients is provided. The method comprises the step ofdetermining ERK8 levels in the cells of a biological sample obtainedfrom said patient, comparing the ERK8 expression in said sample cells toERK8 expression in non-cancer cells, wherein a decrease of greater than20% relative to the non-cancer cell levels is a diagnostic indicationthat the patient has cancer. In one embodiment the ERK8 level to bemeasured is the relative ERK8 protein concentration, in an alternativeembodiment the ERK8 level to be measured is the relative ERK8 kinaseactivity. The ERK8 levels in the patient's biological sample arecompared to a reference “normal” level established for non-cancerouscell. Preferably, the reference sample is based on non-neoplastic cellsof the same tissue type as the biological sample (i.e. biopsy) recoveredfrom the patient. In one embodiment the reference normal ERK8 levels areestablished based on a large population of cells recovered from multiplehealthy individuals. In one embodiment a reduction of greater than 50%in ERK8 levels is indicative of the presence of cancer cells in saidpatient, in another embodiment a reduction of 30%, 40%, 50%, 60%, 70% orgreater is indicative of cancer.

Typically the biological sample used to measure ERK8 levels is recoveredfrom the patient in the form of a tissue biopsy. However, the biologicalsample may be recovered by less intrusive means including drawing bloodfrom the patient and measuring the ERK8 levels of blood cellularcomponents.

As indicated in FIG. 5, ERK8 levels continue to decrease in more severegrades of cancer. According, in one embodiment of the present inventionERK8 levels are used to characterize the aggressiveness or severity ofthe tumor to help determine treatment therapy strategies. The method fordiagnosing and determining the prognosis of a cancer patient comprisesthe step of determining the level of ERK8 expression in a biopsy sample,wherein the severity of the decrease in ERK8 expression is correlatedwith the stage of the cancer. Furthermore, to assist in determining theprognostic outlook and help define therapeutic strategies, estrogenreceptor alpha (ER α) levels in the biological sample can also bemeasured. Increased estrogen receptor alpha (ER α) levels (relative tohealthy population levels) in combination with below normal ERK8 levelsindicate a more advanced stage of cancer.

In a further embodiment ERK8 levels (i.e. either nucleic acid or proteinconcentration or ERK8 kinase activity) or the rate of endogenous ERαdegradation can be monitored as an indicator of the effectiveness of ananticancer therapy. Successful therapy would be indicated by either anincrease in ERK8 levels or by an increase in endogenous ERα degradationduring therapy. Monitoring ERK8 levels in estrogen responsive cancercells cultured in vitro can also be used to identify the effectivenessof anti-cancer agents and help identify new anti-cancer agents. Inaccordance with one embodiment a method of monitoring the effectivenessof an anti-cancer agent for treating estrogen responsive cancers isprovided. The method comprises the steps of monitoring ERK8 levels inestrogen responsive cancer cells contacted with one or more anti-canceragents. The cancer cells may come in contact with the anti-cancer agentin vivo during the course of treating a cancer patient or alternativelythe cancer cells may be culture in vitro and contacted with theanti-cancer agent. In accordance with one embodiment the cancer cells tobe contacted in vitro with the anti-cancer agent are selected fromvarious established tumor cell lines. In another embodiment the cancercells to be contacted in vitro with the anti-cancer agent are recoveredfrom a patient and treated in vitro, as a means of optimizing oridentifying the best anti-cancer therapy.

In one embodiment, the ERK8 levels are determined using antibodiesspecific for ERK8, typically in an ELISA assay, immunohistochemicalanalysis or through use of flow cytometry utilizing antibodies orfluorescent, covalently-bound small molecules which specificallyinteract with ERK8. Polyclonal antibodies raised against rat ERK7 havebeen found to also recognize human ERK8 as shown using recombinant ERK8and by siRNA performed in MCF-10A cells. In accordance with oneembodiment, below average ERK8 protein levels are detected through theuse of a capillary fill device. In one embodiment the capillary filldevice comprises a base provided with a capillary space, said capillaryspace having an interior surface and a first and second end;

a first port formed on an exterior surface of said base and in fluidcommunication with the first end of said capillary space;

a second port formed on an exterior surface of said base and in fluidcommunication with the second end of capillary space;

a reaction zone and a detection zone each formed on said interiorsurface of the capillary space, wherein said reaction zone comprises alabeled ERK8 antibody and the detection zone is located more proximal tothe second port than the reaction zone, wherein fluid introduced intosaid first port will travel through the capillary space, contacting thereaction zone and then the detection zone as the fluid moves to thesecond port. Preferably the ERK8 is deposited on the reaction zone in amanner that contact with fluid moving through the capillary spacereleases the antibody and allows the antibody to specifically bind toany corresponding target antigen present in fluid. Accordingly when afluid derived from a biological sample is placed in the first port thefluid enters the capillary space and resuspends the ERK8 antibody whichthen binds to any ERK8 present in the biological sample fluid. The fluidthen comes in contact with the detection zone wherein the amount of ERK8present in the biological sample derived fluid is determined. In oneembodiment the detection zone comprises a second antibody specific forERK8 (preferably binding an ERK8 epitope separate and distinct from theepitope that the first ERK8 antibody binds), wherein the second antibodyis linked to capillary inner surface and immobilizes any ERK8antibody/ERK8 complexes coming in contact with the second antibody. Inthis embodiment the signal generated by the entrapped labeled ERK8antibody/ERK8 complex corresponds to the concentration of ERK8 withinthe fluid administered to the device.

In one embodiment, the present invention is directed to antibodies thatspecifically bind to ERK7 or ERK8. These antibodies are used to detectERK8 present in human derived biological tissues. In one embodiment,ERK8 antibodies are generated that bind to full length ERK8 but not atruncated mutant of ERK8, such as a polyclonal antibody to the extremeC-terminus of ERK8. In another embodiment, ERK8 antibodies may alsodetect both active and inactive ERK8, such as anti-AL ERK8, whichrecognizes residues in the activation loop of ERK8. In one embodimentthe antibodies are generated to the sequences CRSALGRLPLLPGPRA (SEQ IDNO: 4) and CQALTEY (SEQ ID NO: 5), and in a further embodiment theantibodies are monoclonal antibodies. These antibodies may be used asdiagnostic or therapeutic markers for cancer.

Antibodies to ERK7 and ERK8 polypeptides or peptide fragments thereofmay be generated using methods that are well known in the art. For theproduction of antibodies, various host animals, including but notlimited to rabbits, mice, rats, etc can be immunized by injection withan ERK7/8 polypeptide or peptide fragment thereof. Various adjuvants maybe used to increase the immunological response, depending on the hostspecies, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

For preparation of monoclonal antibodies, any technique which providesfor the production of antibody molecules by continuous cell lines inculture may be used. For example, the hybridoma technique originallydeveloped by Kohler and Milstein (1975, Nature 256:495-497), as well asthe trioma technique, the human B-cell hybridoma technique (Kozbor etal., 1983, Immunology Today 4:72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In anadditional embodiment of the invention, monoclonal antibodies can beproduced in germ-free animals utilizing recent technology(PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact,according to the invention, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for epitopes of ERK8 polypeptides togetherwith genes from a human antibody molecule of appropriate biologicalactivity can be used; such antibodies are within the scope of thisinvention.

According to one embodiment of the invention, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce protein-specific single chain antibodies. Anadditional embodiment of the invention utilizes the techniques describedfor the construction of Fab expression libraries (Huse et al., 1989,Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for sperm surfaceproteins, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, theFab fragments which can be generated by treating the antibody moleculewith papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). Antibodies generated in accordancewith the present invention may include, but are not limited to,polyclonal, monoclonal, chimeric (i.e. “humanized” antibodies), singlechain (recombinant), Fab fragments, and fragments produced by a Fabexpression library.

The antibodies of the present invention can be combined with a carrieror diluent to form a composition. In one embodiment, the carrier is apharmaceutically acceptable carrier. In another embodiment the antibodyis linked to a solid support, and in a further embodiment the antibodyis releasably bound to a solid support, using standard techniques knownto those skilled in the art. For example, the antibody can be simplyadhered to the surface of the support by drying a liquid compositioncomprising the antibody, or alternatively the antibody can be covalentlybound through either an enzymatically cleavable or photoliable linkermoiety or the polypeptide can be bound through ionic interactions andsubsequently released by changes in salt or pH conditions. In anotherembodiment the antibody is linked to a detectable marker.

The ERK7 and ERK8 antibodies of the present invention can be labeledusing standard techniques and detectable markers known to those skilledin the art. The label can be either directly linked or indirectly linkedto the ERK7/8 antibody. In the indirect method, the detectable marker isattached to a secondary antibody that recognizes the ERK7/8 specificmonoclonal antibody. The indirect method has the advantage that it canamplify the signal by binding more of the detectable marker at theantigen site, thus its potential signal on its target may be strongerthan the direct method, especially at low antibody-conjugateconcentrations. A drawback of this method is that it employs twoseparate steps of antibody addition. The direct method has the advantagethat it reduces the number of washing steps and is quicker. The use of asingle labeled immunoreagent also reduces the background by eliminatingnon-specific binding of the secondary antibody.

In accordance with one embodiment the ERK7/8 antibody is labeled andused in a device to measure the amount of ERK7/8 present in a biologicalsample. In one embodiment the ERK7/8 antibody is a monoclonal antibody.Such antibodies have utility as diagnostic markers for cancer as well asutility in therapeutic applications.

In one embodiment, the present invention provides methods of screeningfor agents, small molecules, or proteins that interact with polypeptidescomprising the sequence of SEQ ID NO:1 or a fragment thereof. Theinvention encompasses both in vivo and in vitro assays to screen smallmolecules, compounds, recombinant proteins, peptides, nucleic acids,antibodies etc. which bind to or modulate the kinase activity of ERK7and ERK8. Modulators of ERK7 and ERK8 are anticipated to havetherapeutic use.

In one embodiment of the present invention ERK8, or fragments thereofare used to isolate ligands that bind to ERK8 under physiologicalconditions. The method of isolating such ligands comprises the steps ofcontacting an ERK8 polypeptide with a mixture of compounds underphysiological conditions, removing unbound and non-specifically boundmaterial, and isolating the compounds that remain bound to the ERK8polypeptide. Typically, the ERK8 polypeptide will be bound to a solidsupport, using standard techniques, to allow for rapid screening ofcompounds. In one embodiment the ERK8 polypeptide is releasably bound toa solid support, using standard techniques known to those skilled in theart. For example, the polypeptide can be covalently bound through eitheran enzymatically cleavable or photoliable linker moiety or thepolypeptide can be bound through ionic interactions and subsequentlyreleased by changes in salt or pH conditions. The solid support can beselected from any surface that has been used to immobilize biologicalcompounds and includes but is not limited to polystyrene, agarose,silica or nitrocellulose. In one embodiment the solid surface comprisesfunctionalized silica or agarose beads. Screening for such compounds canbe accomplished using libraries of pharmaceutical agents and standardtechniques known to the skilled practitioner.

Ligands that bind to the ERK7 and/or ERK8 polypeptides can then befurther analyzed for agonists and antagonists activity through the useof an in vitro kinase assay such as that described in Example 2.Compounds that enhance the activity of ERK8 have potential use as agentsfor treating cancers that are responsive to estrogen. These include, butare not limited to, breast, ovarian, prostate, endometrial, colon,pancreas and brain cancers and neoplastic tumors. In one embodimentcompounds are screened for their ability to modulate ERK7 and/or ERK8kinase activity, and in one embodiment compounds are screened for theirability to enhance ERK8's kinase activity.

In accordance with one embodiment, a method for identifying modulatorsof ERK7/8 activity is provided. The method comprises the steps of

contacting an ERK7/8 polypeptide with a kinase substrate, and ATP, toform a control mixture;

contacting an ERK7/8 polypeptide with a kinase substrate, ATP, and apotential ERK7/8 activity modulator, to form a reaction mixture;

incubating the control and reaction mixtures under identical conditionsfor a predetermined length of time; and

comparing the amount of phosphorylated substrate produced in thereaction mixture to that produced in the control mixture, wherein adifferent concentrations of phosphorylated substrate produced by thecontrol and reaction mixtures identifies a ERK7/8 activity modulator. Inaccordance with one embodiment compounds are screened for their abilityto enhance ERK7/8's ability to phosphorylate its substrate relative toan ERK7/8 kinase reaction run in the absence of the compound. In anotherembodiment compounds are screened for their ability to inhibit ERK7/8'sability to phosphorylate its substrate relative to an ERK7/8 kinasereaction run in the absence of the compound. Compounds that enhanceERK8's kinase activity can be formulated as pharmaceutical compositionsand administered to a subject to treat cancer and neoplastic tumors forestrogen responsive neoplastic growths. In advanced stage and aggressivecancers ERα may be at such low levels that treatment with an ERK8 kinaseinhibitor may be advantageous to maintain the cancer in a state that isresponsive to anti-hormonal therapy.

Example 1

Erk7 Specifically Enhances ERα Destruction

Materials and Methods

Reagents

The monoclonals, 12CA5 (anti-HA) and 9e10 (anti-MYC), were obtained fromthe Univ. Virginia Lymphocyte Culture Facility. The anti-active MAPKantibody (also called anti-pTEY antibody) was purchased from Promega.The cDNAs for human ERα, human IκBα, human AR, and mouse SF1 wereprovided by P. Chambon (EMBO J. 8:1981-1986.), D. Ballard (Mol CellBiol. 15:2809-2818), M. Weber (J Biol Chem. 277:29304-29314) and K.Parker (Mol Endocrinol. 9:1233-1239), respectively. MEKc was provided byN. Ahn (J Biol. Chem. 267:25628-25631), ERK5 by J. Dixon (J Biol Chem.270:12665-12669) and HAubiquitin by D. Bohmann (Cell. 78:787-798). Thepeptides to the C-terminal end of rat ERK7 (CRSALGRLPLLPGPRA; SEQ ID NO:4) and to the activation loop of rat ERK7 (CQALTEY; SEQ ID NO: 5) andthe polyclonals, anti-ERK7 and anti-AL ERK7, were produced by ResearchGenetics.

Expression Vectors

The cDNA for ERK7 was obtained from a rat testis cDNA library using thepolymerase chain reaction (PCR) with primers specific to the publishedERK7 sequence (Mol Cell Biol. 19:1301-1312). The sequence was verifiedand the cDNA was inserted in frame into pK3H (contains a triple HA tag)or PKR7-MYC (contains a MYC tag) or modified pCMV-Tag5 (Stratagene). ThecDNA for ERK8 was obtained from a human EST library. The sequence wasverified and the cDNA was inserted in frame into pK3H or PKR7-MYC ormodified pCMV-Tag5. The vector pCMV-Tag5 (Stratagene) was modified byinserting the promoter and triple HA tag sequences from pKH3 intopCMV-Tag5. All ERα and ERK7 and ERK8 mutants were produced using PCR andthe sequences verified by the Univ. Virginia Biomolecular ResearchFacility. Both w.t. and mutant ERαs were sub-cloned into pSG5.

Immunoblots

BHK cells were transfected in 10 mm dishes with calcium phosphate and 1μg of wild type or mutant ERα or FLAG-IκBα or HA-SF1 or HA-AR construct,5 μg of a construct encoding a wild type or mutant kinase or vectorcontrol. At 8 h post transfection, the cells were washed withphosphate-buffered saline (PBS) and placed in Dulbecco's modifiedEagle's medium (DMEM) with 5% fetal calf serum (FCS). After 1 h thecells were washed with PBS, serum starved for 15 h and either estradiolor vehicle added 6 h before lysis with boiling SDSPAGE sample buffer(−DTT, see Joel et al., (1998) J Biol Chem. 273:13317-13323). For therate experiments transfected cells were trypsinized, aliquoted andpre-treated with 50 μM cycloheximide for two hours in the absence ofserum before the addition of estradiol. The cells were lysed at varioustimes after estradiol treatment.

Breast lines were serum-starved for 15 h before lysis with sample buffer(−DTT). Human tissue obtained from the University of Virginia TissueProcurement Facility was pulverized in the presence of liquid N₂ and thepowder added to sample buffer (−DTT). Total protein was determined usingDC protein assay (Bio-Rad) and 100 mg of protein electrophoresed (DTTwas added to each sample prior to loading) and transferred tonitrocellulose. Immunoblots and densitometric analysis were as described(J Biol Chem. 273:13317-13323).

Immunoprecipitations

BHK cells were transfected in 150 mm dishes with calcium phosphate and2.5 μg of a ubiquitin construct and either 22.5 μg of MYC-ERK7 or vectorcontrol. Additionally, the cells were co-transfected with 2.5 μg ofMYC-ERα(282-595) or additional vector control. The transfected cellswere washed and then serum starved. Lysis and immunoprecipitation with12CA5 were as described (EMBO J. 20:3484-494).

Generation of Stable Clones

MCF-7 cells were maintained in DMEM with 5% charcoal-stripped (cs) FCSand 5% CO₂. They were transfected with Lipofectamine (Life Technologies)according to the manufacturer's directions with 6 μg of the modifiedpCMV-Tag5 vector with or without the K43A-ERK7 cDNA or ERK8 cDNA. Stableclones were obtained using G418 (600 mg/ml) selection. Isolated colonieswere cloned, propagated and where appropriate the lines were analyzedfor expression of HA-K43A-ERK7.

Transcriptional Analysis

MCF-7 stables were transfected and assayed for luciferase andβ-galactosidase as described previously (EMBO J. 20:3484-494).

Proliferation

MCF-7 stables were seeded at 5×10⁴ in a 24 well dish containing DMEM and5% csFCS with 600 μg/ml G418. After 15 hrs the cells were treated withor without 1 μM ICI 182,780. At various times the cells were lysed andthe amount of ATP determined by CellTiter-Glo™ Luminescent CellViability Assay according to the manufacturer's protocol (Promega).

Results

Erk7 Specifically Enhances ERα Destruction

To test whether members of the MAPK(ERK1/2) family regulate ERαturnover, family members were investigated for their ability toinfluence the steady state levels of ERα expression. ERα and theindicated kinases were co-expressed in baby hamster kidney (BHK) cellsand the expression levels of ERα and the kinases determined byimmunoblotting the lystates of the transfected cells. In theseexperiments BHK cells were chosen because they are easily transfectedand support ERα-mediated transcription when provided with ERα cDNA.Ectopic expression of MAPK(ERK1/2) would not substantially increasecellular MAPK(ERK1/2) activity because in order for MAPK(ERK1/2) to beactive it must be phosphorylated by MAPK kinase (MEK). Therefore, aconstitutively active mutant of MEK (MEKc) was expressed to activateendogenous MAPK(ERK1/2). In this transient transfection system we didnot observe any difference in the steady state levels of ERα betweenvehicle and estradiol treatments in the vector control (FIG. 8).However, in the vector control, the amount of ERα present in the slowerelectrophoretic migrating band increased in the presence of estradiol.We previously determined that phosphorylation of Ser-118 in human ERαresults in a reduced electrophoretic mobility. Activation of endogenousMAPK(ERK1/2) by MEKc increased the amount of ERα present in the slowermigrating band, which can be most clearly observed by comparison withthe vector control in the absence of estradiol. These data areconsistent with observations that MAPK(ERK1/2) phosphorylates Ser-118.

The antipTEY antibody (also referred to as the anti-active MAPKantibody) specifically recognizes the dual-phosphorylated Thr and Tyrresidues present in the activation loop of MAPK(ERK1/2) and ERK7. Thus,as expected, the anti-pTEY immunoblot shows that the expression ofHA-MEKc enhanced the activity of MAPK(ERK1/2) above that observed withthe vector control. However, increasing MAPK(ERK1/2) activity did notalter ERα protein levels compared to the vector control. Remarkably,however, the ectopic expression of ERK7 decreased the steady statelevels of ERα expression ˜2-fold in the absence and ˜3-fold in thepresence of estradiol (FIG. 8). The immunoblot with the anti-pTEYantibody demonstrates that similar levels of active ERK7 and endogenousMAPK(ERK1/2) were achieved. Expression of another member of the TEYkinase subgroup, ERK5, did not influence ERα levels, furtherdemonstrating that a decrease in the steady state level of ERα is aspecific response to ERK7.

Steady state levels reflect both the rates of synthesis and degradation.Therefore, to determine the effect of ERK7 on the rate of ERαdestruction we blocked protein synthesis using the inhibitor,cycloheximide, and determined the amount of ERα by densitometry atvarious times after the addition of vehicle or estradiol. To eliminatetransfection differences between time points the BHK cells weredistributed into the appropriate number of plates after beingtransfected. The rate data were fitted using a single exponential decayand the rate constants determined. Based on these rate constants, thet1/2 for ERα in BHK cells is ˜4 hrs and in the presence of estradiol thet1/2 is ˜3 hrs. In BHK cells ectopic expression of ERK7 decreases thet1/2 to ˜2.5 hrs and ˜1.9 hrs in the absence and presence of estradiol,respectively. These data also demonstrate that the decrease in ERαlevels in the presence of ERK7 is not a transfection artifact becausethe rate data are independent of transfection efficiency.

To understand the role that the kinase activity of ERK7 plays inenhancing ERα destruction the ability of the mutant K43A-ERK7 toinfluence ERα steady state levels was determined. In this mutant theessential Lys-43, necessary for ATP hydrolysis, has been changed to Ala.K43A-ERK7 does not have catalytic activity in an in vitro kinase assayusing myelin basic protein as a substrate, in agreement with Abe et al.(Mol Cell Biol. 19:1301-1312) who found that mutating Lys-43 to Argdestroyed ERK7 catalytic activity. Co-expression of ERα with kinase-deadERK7 (HA-K43A-ERK7) did not significantly decrease ERα protein levelscompared to the vector control (FIG. 8). Similar effects were alsoobserved with a kinase-inactive ERK7 in which the Thr and Tyr present inthe TEY phosphorylation motif were mutated to Ala. Wild type andkinase-dead ERK7 were expressed to similar extents but only wild typeERK7 is able to decrease ERα levels. Therefore, these data demonstratethe kinase activity of ERK7 is required to decrease ERα protein levels.

ERK7 Specifically Enhances ERα Degradation.

ERα is degraded through the 26S proteasome pathway, and one possibleexplanation for the observed decrease in ERα levels is that ERK7enhances the rate of proteasome-mediated degradation. Therefore, theability of the competitive proteasome inhibitor, MG132, to prevent ERK7from decreasing ERα levels was tested. In agreement with the results ofNawaz et al., addition of MG132 increased ERα protein levels in both thepresence and absence of estradiol. Treatment with MG132 also increasedthe amount of ERα co-expressed with ERK7, and increased the abundance ofhigher MW forms of ERα induced by ERK7. In the presence of a proteasomeinhibitor the ubiquitinated products accumulates and thus the higher MWERα forms are likely due to ubiquitination. Similar results wereobserved with another proteasome inhibitor, lactacystin. It is notpossible to determine by immunoblotting whether these forms are a resultof ubiquitination because anti-ubiquitin antibodies are extremelyinsensitive. Additionally, our anti-ERα antibody does notimmunoprecipitate the higher MW ERα forms. These results suggest thatERK7 decreases the abundance of ERα by enhancing its degradation throughthe proteasome pathway. To test the specificity of ERK7-mediateddegradation we determined whether ERK7 could enhance the destruction ofIκBα, which is degraded by the 26S proteasome pathway. ERK7 did notreduce the protein levels of IκBα in comparison to the vector control.These results support the hypothesis that ERK7 is a specific regulatorand is not a general activator of the ubiquitination/proteasomemachinery.

To determine whether ERK7 can trigger the destruction of other nuclearreceptor superfamily members, ERK7's effect on steroidogenic factor I(SF1), a distant, evolutionarily-related relative of ERα, wasinvestigated. ERK7 had no effect on the protein levels of SF1 comparedto the vector control (FIG. 9A). These results suggest that ERK7stimulates the degradation of only a subset of the nuclear receptorsuperfamily, which includes ERα. To further define the nuclear receptorsthat are targets of ERK7 we tested the ability of ERK7 to enhance therate of degradation of the androgen receptor (AR) a close relative ofERα. The rate of degradation was determined in the presence ofcycloheximide in a similar manner to that performed with ERα, exceptthat dihydryoxytestosterone was used. Remarkably, ERK7 did not enhancethe degradation of the AR (FIG. 9B). Taken together, these data suggestthat ERK7 specifically regulates ERα turnover.

Erk7 is Highly Expressed in Normal Human Breast Cells.

To understand the importance of ERK7 in regulating estrogen action ERK7expression in breast cells, an estrogen target tissue, was investigated.A polyclonal antibody was generated to the extreme C-terminus of ratERK7, which was able to specifically detect ERK7 but not a truncatedmutant of ERK7 in lysates from transfected BHK cells. Deletion of theC-terminal tail of ERK7 also inhibits its constitutive kinase activity,and the truncation is therefore not detected by the anti-pTEY antibody.The anti-ERK7 antibody was used to immunoblot lysates of MCF-10A, anormal breast cell line and the breast cancer cell lines, MCF-7 andMDA-MB-231 (FIG. 10A). A band of ˜60 kDa was strongly detected by theanti-ERK7 antibody in MCF-10A cells and this molecular weight (MW) is inagreement with the calculated MW based on the rat ERK7 cDNA. The ˜60 kDaband recognized by anti-ERK7 co-localized with a band recognized by theanti-pTEY antibody (FIG. 10A). Together, these data strongly suggestthat the ˜60 kDa band observed in normal breast cells is human ERK8. Itis not particularly surprising that the antibody raised to rat ERK7recognizes human ERK8 because a number of kinases are virtuallyidentical in their amino acid sequences between rats and humans, eg.ERK2, and as demonstrated in FIG. 3 rat ERK7 and human ERK8 share a highdegree of sequence similarity.

The MCF-10A cell line has higher levels of ERK7 than the MCF-7 andMDA-MB-231 cell lines. The amount of ERα in the different cell lines isinversely correlated with the amount of ERK7. The amount of ERα isextremely low in MCF-10A and can only be detected by immunoblottinganti-ERα immunoprecipitations. These results are consistent with theidea that ERK7 regulates ERα turnover, because the MCF-10A cell line haslow levels of ERα and the MCF-7 cell line has high levels of ERα. TheMDA-MB-321 cell line represents an intermediate between MCF-10A andMCF-7. To further understand the physiological significance of ERK7 inregulating estrogen action the expression levels of ERK7 in normal humanbreast tissue and breast cancer tissue was determined (see FIG. 10B andFIG. 4). Detection of the 60 kDa band in normal human breast tissue wasblocked by pre-incubating the anti-ERK7 antibody with the antigenicpeptide. In total 13 normal tissues, 5 benign tumors, and 66 breasttumors were examined and the results shown are representative. In FIG. 5the data is presented by grouping the samples according to their tumorgrade from the least to the most aggressive according to thepathologist's report. The tissue samples were normalized to each otherby immunoblotting for Ran, a housekeeping protein whose expression levelis not known to change with any disease state.

ERK7 was expressed in all of the normal and benign tissue samples, ˜halfof the grade 1 samples, ˜20% of the grade 2 tumors, none of the grade 3tumors and ˜30% of the metastatic tumors (FIG. 10B). Thus loss of ERK7is correlated with breast cancer progression. These samples were alsoanalyzed for active MAPK(ERK1/2) and in agreement with the literatureMAPK(ERK1/2) activity was generally higher in breast cancers than in thenormal or benign tumor samples. But, importantly, a correlation betweenMAPK(ERK1/2) activity and loss of ERK7 was not observed. These resultsdemonstrate that the loss of ERK7 is not merely a reflection of ageneral decrease in the levels of various members of the MAPK(ERK1/2)family. Also in agreement with the literature normal human breastmammary epithelial cells were observed to have very low levels of ERα,which is consistent with the data presented herein that normal breasttissue has significant amounts of ERK7. Additionally, cancer tissuesthat had detectable amounts of ERα had undetectable levels of ERK7expression. Thus loss of ERK7 correlates with increased ERα levels.These data further suggest that ERK7 is an important regulator ofestrogen action in the breast.

ERK7 Regulates Hormone Responsiveness in Human Breast Cells

The concentration of ERα is limiting for estrogen responsiveness invivo, and ERα synthesis and degradation, therefore, play a pivotal rolein controlling the expression of ERα-regulated genes. Taken together,the present results indicate that ERK7 regulates hormone responsivenessin cells that endogenously express ERα. To test this hypothesis, thecreation of MCF-7 clones that stably expressed HA-ERK7 was attempted,however, the majority of the synthesized ERK7 was not active. Theseresults suggest that ERK7 activity is regulated differently in breastcells than in BHK cells in which ˜50% of ERK7 is active. Kinase-deadmutants are often able to act as a dominant negatives and therefore,MCF-7 clones were produced that stably expressed K43A-ERK7. Thehypothesis was that the MCF-7 cells contained extremely low levels ofERK7 and that the cell's ability to degrade ERα be inhibited by usingK43A-ERK7. In MCF-7 lysates a faint band at ˜60 kDa was observed uponextended exposure of the anti-ERK7 immunoblot. The anti-ERK7 antibody isable to immunoprecipitate HA-ERK7 from lysates of transfected cells andtherefore, this antibody was used to immunoprecipitate ERK7 from MCF-7lysates. ERK7 was detected in the inimunoprecipitate using anotherpolyclonal antibody to ERK7 that recognizes residues in the activationloop of ERK7 (anti-AL ERK7). The anti-AL ERK7 recognizes both the activeand inactive forms of ERK7. Thus these results demonstrate that MCF-7cells do contain ERK7. In the K43A-ERK7 stable cell lines, addition ofcycloheximide decreased the degradation of ERα by ˜1.5 fold and >4 foldin the absence and presence of estradiol, respectively, compared to thatin the vector stable lines. Thus K43A-ERK7 is more effective atinhibiting the degradation of the hormone-bound receptor than that ofthe unbound receptor. Over the 4 hr cycloheximide treatment, the levelof K43A-ERK7 decreased significantly, which suggests that ERK7 is turnedover rapidly in MCF-7 cells. It is likely that the effects on ERαdegradation would have been much more dramatic had the levels ofK43AERK7 remained constant over the time course of the experiment. Theseresults suggest that K43A-ERK7 is able to act as a dominant negative inMCF-7 cells by inhibiting the action of endogenous ERK7.

To further test whether ERK7 is an important regulator of estrogenaction the effect of K43A-ERK7 on ERα-regulated transcription wasinvestigated. The MCF7 stable lines were cotransfected with a vectorencoding the luciferase reporter under the control of estrogenresponsive elements (ERE), plus a control vector encodingβ-galactosidase. Cells expressing K43A-ERK7 had ˜3 fold greatertranscriptional response to estradiol than the control cells. MG132 alsoincreased the transcriptional response of the control cells by ˜3 fold.However, the transcriptional response in the K43A-ERK7 cells was notaffected by MG132. These results suggest that K43A-ERK7 and MG132influence ERα-mediated transcription through a similar mechanism, byinhibiting the ERα degradation rate. In these experiments, a TATA boxand a dual ERE regulates luciferase reporter expression. Therefore, ERK7was tested to determine whether ERK7 also regulates ERα-regulatedtranscription of endogenous genes in MCF-7 cells. As a readout for broadresponses to ERα the ability of estrogen to stimulate MCF-7 cellproliferation was used because the proliferation response is known to beregulated by ERα transcriptional activation. It is likely that ERK7regulates the activity of molecules in addition to ERα and these othermolecules may influence proliferation. Therefore, to examine only theeffects of K43A-ERK7 on ERα-mediated proliferation growth was determinedin the presence of vehicle or ICI 182,780, a pure estrogen antagonist.To examine only the ERα-dependent growth the growth in the presence ofICI 182,780 was subtracted from that obtained with the vehicle.K43A-ERK7 enhanced the rate of ERα-dependent proliferation ˜2-fold (FIG.11). These data suggest that ERK7 regulates ERα-mediated transcriptionfrom both simple and complex promoters. Furthermore, the ability ofK43A-ERK7 to promote proliferation suggests that ERK7 regulation of ERαdegradation rate is important in determining estrogen responsiveness.

ERK7 Phosphorylation of ERα does not Influence ERα Stability

Because turnover is commonly regulated by phosphorylation of the targetprotein, it seemed possible that ERK7 regulates ERα degradation bydirect phosphorylation. ERK7 is most likely a proline-directedserine/threonine kinase because the catalytic domain is highly relatedto MAPK(ERK1/2) and thus there are four putative ERK7 phosphorylationsites in ERα, Ser-104, Ser-106, Ser-118 and Ser-294. It has beenpreviously determined that Ser-118 is the major site of phosphorylationin response to estradiol binding and therefore, whether ERK7 couldphosphorylate ERα in vivo was investigated. Lysates from cellstransfected with wild type ERα or Ser-118A-ERα in the presence orabsence of ERK7 were normalized for total ERα and immunoblotted withanti-ERα. The lysates were normalized to ERα rather than total proteinso that the intensities of the various ERα bands between the ERK7 andvector control samples could be directly compared.

The appearance of Ser-118 phosphorylation can be observed as a sharpband and ERK7 enhances the amount of this phosphorylation in both theabsence and presence of estradiol. The kinase dead mutant, K43A-ERK7,diminishes the intensity of this slower migrating ERα band. Takentogether, these data suggest that ERK7 can regulate-either directly orindirectly—the level of Ser-118 phosphorylation. ERK7 also increases thenumber of other higher MW ERα forms that are observed as diffuse bandsboth in wild type ERα and the S118A-ERα mutant.

To determine the role that ERα phosphorylation may play in ERK7-mediatedERα destruction the ability of ERK7 to enhance the rate of S118A-ERαdegradation in the presence of cycloheximide was determined. ERK7 wasable to enhance the degradation of S118A-ERα with a t1/2 similar to thatobserved with the wild type ERα. ERK7 was also tested to determinewhether ERK7 could enhance the degradation of mutant ERαs that containedmutations in the other putative ERK7 phosphorylation, Ser-104, Ser-106and Ser-294. There were no significant differences in the ability ofERK7 to decrease wild type ERα protein levels compared to any of themutants. ERK7 also did not enhance the degradation of a deletion mutantof ERα lacking the ligand-binding domain (ERα (1-282). These data areconsistent with those obtained with MEKc in which enhanced Ser-118phosphorylation by MAPK(ERK1/2) did not influence ERα levels. Thusalthough ERK7 may enhance Ser-118 phosphorylation it seems thatmechanisms other than ERα phosphorylation are important in targeting ERαfor destruction.

The ligand-Binding Domain Targets ERα for ERK7-Mediated Destruction

ERK7 targets the wild type ERα for degradation but not a deletion mutantlacking the ligand-binding domain. Therefore, ERK7 was investigated todetermine whether it enhanced the destruction of the ligand-bindingdomain. In agreement with the literature, estradiol was observed toenhanced the level of ERα (283-595) co-expressed with the vectorcontrol. Nonetheless, ERK7 was able to decrease the expression level ofERα (283-595) in the presence and absence of estradiol. These resultssuggest that the ligand-binding domain plays an important role in theability of ERK7 to regulate ERα protein levels. Inhibitors of the 26Sproteasome pathway are able to decrease ERK7-mediated ERα degradationand ERK7 enhances the formation of higher ERα MW forms. Therefore, it isanticipated that the high MW forms consist of ubiquitinated ERα.

However, it is not possible to directly determine whether these higherMW forms are the result of ubiquitination. Therefore, to test thehypothesis that ERK7 enhances ERα ubiquitation, BHK cells wereco-transfected with constructs encoding HA-ubiquitin plus eitherMYC-tagged ERα(283-595) or vector control. Additionally, the cells weretransfected with either MYC-tagged ERK7 (MYC-ERK7) or vector control andtreated with MG132. To isolate ubiquitinated proteins, lysates wereimmunoprecipitated with anti-HA antibody, then immunoblotted with eitheranti-MYC or Ab10, an antibody to the ligand-binding domain of ERα. Theamount of ERα(283-595) containing HA-ubiquitin was greatly increased inthe presence of ERK7 compared to the vector control. Bands wereidentified at a MW consistent with the addition of a single ubiquitin toMYC-ERα(283-595), whereas the other bands were observed consistent withpolyubiquitination. The results with Ab10 were similar to those obtainedwith the α-MYC antibody). These results support the hypothesis that ERK7enhances the level of ERα ubiquitination.

Discussion

It has been previously reported that, in response to hormone, thephosphorylation of Ser-118 increases. ERK7 enhances Ser-118phosphorylation in vivo but this phosphorylation is not important forERK7-mediated degradation because ERK7 is able to enhance the rate ofdegradation of ERα and S118-ERα to the same extent. In addition, noevidence was uncovered that mutation of other putative ERK7phosphorylation sites influenced ERK7-mediated ERα degradation. It seemsunlikely that phosphorylation of the ligand-binding domain plays a rolein regulating turnover because enhanced phosphorylation in theligand-binding domain has not been observed using (³²P)-orthophosphatelabeling and microsequencing of the radiolabeled ERα peptides in theabsence or presence of lactacystin.

There are several cases known in which the destruction of specifictargets is regulated by kinases. The kinases phosphorylate a residuewithin a specific sequence context, and this phosphorylation results inthe interaction with particular E2 and E3 enzymes. For example, glycogensynthase kinase −3β phosphorylates β-catenin and IκB kinasephosphorylates IκBα, which regulates their destruction through theSkp1-Cullin-F-box complex. There is also evidence that the destructionof some members of the nuclear steroid receptor superfamily is regulatedby their phosphorylation. However, the discovery of ERK7-mediateddestruction of ERα is mechanistically distinct. One possible mechanismis that ERK7 phosphorylates a component of the ubiquitin machinery,which increases its catalytic activity or affinity for ERα. Anotherplausible mechanism is that ERK7 phosphorylates an ERα-interactingprotein, which exposes a surface on ERα that targets it for destruction.

Remarkably, ERK7 expression is lost during breast cancer progression.Normal breast epithelia cells have extremely low levels of ERα and areconsidered to be ERα-negative. Hormone-responsive breast cancers express10-fold or more higher ERα levels but as the cancer becomes moreaggressive ERα expression is frequently lost. In our studies all the ERαpositive tumors also had lost ERK7 expression and it may be that theincrease in ERα levels in hormone responsive breast cancers is due todecreased ERK7 expression. However, not all tumors that had lost ERK7expression were ERα positive, suggesting that other factors in additionto ERK7 are involved in regulating ERα levels. It may be that duringtumor progression ERK7 downstream effectors have been inappropriatelyactivated, resulting in reduced ERα levels even in the absence of ERK7.Accordingly it is believed that ERK7 is important in maintaining thehomeostasis of a normal breast cell and that with high frequency ERK7 islost at an early step in breast cancer progression.

Example 2

Assay for Measuring ERK7 & 8 Activity

Kinase assays were developed to measure the activity of rat ERK7 andhuman ERK8. Purified, recombinant ERK7 and ERK8 were obtained byintroducing a HIS-tag in frame with the coding sequence of the kinases,expressing the proteins in bacteria and purifying the proteins over aNiNTA resin (Qiagen, Valencia, Calif.). Immunoprecipitated ERK7 and ERK8were obtained by introducing a HA-tag in frame with the coding sequenceof the kinases, expressing the proteins in baby hamster kidney cells andimmunoprecipitating and eluting the kinases off the beads using anHA-peptide.

The kinase assays were performed in a 96 well format according to thefollowing procedure. Glutathione-S-transferase (GST)-fusion protein (1μg/well) containing the sequence—KNIVTPRTPPPSQGKG (SEQ ID NO: 6;corresponding to human myelin basic protein) (GST-MBPtide) was adsorbedin the wells of LumiNunc 96-well polystyrene plates (MaxiSorp surfacetreatment). The wells were blocked with sterile 3% tryptone in phosphatebuffered saline and stored at 4° C. for up to 6 months. ERK7 or ERK8 (5nM of purified, bacterially expressed or kinase immunoprecipitated fromapproximately 10% of a confluent 15 cm tissue culture plate) in 70 μl ofkinase buffer (5 mM β-glycerophosphate pH 7.4, 25 mM HEPES pH 7.4, 1.5mM DTT, 30 mM MgCl₂, 0.15 M NaCl) was dispensed into each well.Reactions were initiated by the addition of ATP and terminated after 30to 60 min by addition of 75 μl 500 mM EDTA, pH 7.5. All assays measuredthe initial velocity of reaction. After extensive washing of wells,monoclonal phosphospecific antibody developed against the phosphopeptide(anti-Phospho MBP, Upstate Biotechnology, catalog # 05-429) andHRP-conjugated anti-mouse antibody (115-035-146, Jackson ImmunoResearchLaboratories, West Grove, Pa.) were used to detect threoninephosphorylation of the substrate. HRP activity was measured usingWestern Lightning Chemiluminescence Reagent (NEL102, PerkinElmer LifeSciences) according to manufacturer's protocol. Maximum and minimumactivity is the relative luminescence detected in the presence ofvehicle and 200 mM EDTA, respectively.

The kinase assays were characterized with respect to concentration ofERK7 and ERK8 required, time of incubation and concentration of ATPrequired. FIG. 1 demonstrates the K_(M) of Mn⁺⁺-ATP for bacteriallyexpressed ERK7. The K_(M) is approximately 100 μM. Also shown in FIG. 1is the high signal:background ratio that can be achieved by this assay.Kinase activity in the presence of 1 mM ATP of the recombinant ERK7 andERK8 and immunoprecipitated ERK7 is depicted in FIG. 2. Thus, the assaysare suitable for measuring activity of ERK7 and ERK8.

1. A method of diagnosing the presence of an estrogen responsive cancerin a patient, said method comprising the step of measuring ERK8 levelsin the cells of a biological sample obtained from said patient; anddetermining if the ERK8 levels of the biological sample aresignificantly lower than those detected in non-cancerous cells, whereinsignificantly lower ERK8 levels indicates the presence of cancer cellsin said patient.
 2. The method of claim 1 wherein a decrease of greaterthan 20% in the ERK8 levels of the biological sample relative to thenon-cancer cell levels indicates the presence of cancer cells in saidpatient.
 3. The method of claim 2 wherein the estrogen responsive cancerto be diagnosed is selected from the group consisting of breast, ovarianand endometrial cancers.
 4. The method of claim 3 wherein the estrogenresponsive cancer to be diagnosed is breast cancer.
 5. The method ofclaim 1 wherein the ERK8 level measured is the concentration of the ERK8protein.
 6. The method of claim 1 wherein the ERK8 level measured is theERK8 kinase activity.
 7. A method for diagnosing and determining theprognosis of a cancer patient, said method comprising the step ofdetermining ERK8 levels in a biopsy sample, wherein the severity of thedecrease in ERK8 levels is correlated with more advanced stages of thecancer.
 8. The method of claim 7 further comprising the step ofdetermining estrogen receptor alpha (ER α) levels in the biopsy sample,wherein increased estrogen receptor alpha (ER α) levels (relative tohealthy population levels) in combination with below normal ERK8 levelsindicates a more advanced stage of cancer.
 9. An antibody thatspecifically binds to the amino acid sequence of SEQ ID NO:
 1. 10. Theantibody of claim 9 wherein the antibody is labeled.
 11. The antibody ofclaim 10, wherein the antibody is a monoclonal antibody.
 12. Theantibody of claim 11 wherein the antibody is releasably bound to a solidsupport.
 13. A device for measuring ERK7/8 levels, said devicecomprising a base provided with a capillary space, said capillary spacehaving an interior surface and a first and second end; a first portformed on an exterior surface of said base and in fluid communicationwith the first end of said capillary space; a second port formed on anexterior surface of said base and in fluid communication with the secondend of capillary space; a reaction zone and a detection zone each formedon said interior surface of the capillary space, wherein said reactionzone comprises a labeled ERK7/8 antibody and the detection zone islocated more proximal to the second port than the reaction zone, whereinfluid introduced into said first port will travel through the capillaryspace, contacting the reaction zone and then the detection zone as thefluid moves to the second port.
 14. The device of claim 13 wherein saiddetection zone comprises a secondary antibody specific for said ERK7/8antibody.
 15. A method of monitoring the effectiveness of an anti-canceragent for treating estrogen responsive cancers, said method comprisingthe steps of monitoring ERK8 levels in estrogen responsive cancer cellscontacted with said agent.
 16. The method of claim 15 wherein the cancercells are contacted in vitro as a means of identifying new anti-canceragents.
 17. The method of claim 16 wherein the cancer cells are selectedfrom various established tumor cell lines.
 18. The method of claim 15wherein the cancer cells are recovered from a patient and treated invitro.
 19. A method for identifying modulators of ERK7/8 activity, saidmethod comprising the steps of contacting an ERK7/8 polypeptide with akinase substrate, and ATP, to form a control mixture; contacting anERK7/8 polypeptide with a kinase substrate, ATP, and a potential ERK7/8activity modulator, to form a reaction mixture; incubating the controland reaction mixtures under identical conditions for a predeterminedlength of time; and comparing the amount of phosphorylated substrateproduced in the reaction mixture to that produced in the controlmixture, wherein a different concentrations of phosphorylated substrateproduced by the control and reaction mixtures identifies a ERK7/8activity modulator.
 20. The method of claim 19 wherein the kinasesubstrate is a polypeptide comprising the sequence of SEQ ID NO: 6.